G484S Amino Acid Substitution in Lanosterol 14-α Demethylase ( ERG11 ) Is Related to Fluconazole Resistance in a Recurrent Cryptococcus neoformans Clinical Isolate

Abstract

Five sequential Cryptococcus neoformans isolates recovered from an AIDS patient with recurrent meningitis were analyzed. Four isolates were fluconazole susceptible, while the fifth isolate developed fluconazole resistance. Analysis of the 14-␣ lanosterol demethylase gene (ERG11) showed a point mutation in the resistant strain responsible for the amino acid substitution G484S.Mechanisms of azole resistance already described for yeasts include altered affinity of lanosterol 14-␣ demethylase (ERG11) to azole drugs due to target site mutation or its overexpression and decreased accumulation of drugs due to enhanced energydependent drug efflux (5, 13). Changes in the azole affinity of the lanosterol 14-␣ demethylase have already been related to low-level fluconazole resistance in Cryptococcus neoformans isolates (15). In addition, the decreased affinity of lanosterol 14-␣ demethylase for azole derivatives due to mutations that contributes to the increase in the MICs of fluconazole has been described for sequential clinical isolates of Candida albicans (5, 6). To elucidate if this mechanism could also be implicated in the resistance of C. neoformans to azole, we compared the ERG11 genomic sequence in five sequential isolates recovered from recurrent episodes of cryptococcal meningitis.Clinical case. The five strains of C. neoformans were isolated from a 33-year-old male patient who had been positive for human immunodeficiency virus since 1990 and was presenting advanced AIDS (CD4 ϩ count, Ͻ100 cells/mm 3 ) and recurrent cryptococcosis. The first episode of cryptococcosis was diagnosed in August 1997 at Hospital Fernandez, Buenos Aires, Argentina. During the following 15 months, four more episodes of cryptococcal meningitis were detected and documented by cultures recovered from cerebrospinal fluid. In the first episode the patient was treated with amphotericin B (AMB), and for the rest of the episodes a fluconazole therapy at different doses was always established, reaching a cumulative dose of 336 g at the moment of the fifth episode. The five isolates from each episode were identified as C. neoformans var. grubii by the following parameters: morphology, assimilation and fermentation of carbon and nitrogen compounds, and molecular taxonomy.Susceptibility testing was performed by microdilution and E-test methods. Candida parapsilosis ATCC 22019 and Candida krusei ATCC 6258 were used as quality control strains throughout the experiments (7). Microdilution method. The susceptibility testing followed the NCCLS recommendations (7) but included some modifications as previously described (11). Briefly, the susceptibility testing included RPMI medium supplemented with 2% glucose as the assay medium (RPMI-2% glucose), an inoculum size of 10 5 CFU/ml, flat-bottomed trays, spectrophotometric reading at 530 nm, incubation at 30°C, and shaking at 350 rpm for 48 h (11). The antifungal agents used in the study were as follows: AMB (Sigma Aldrich Quimica S.A., Madrid, Spain), 5-flucytosine (5FC) (Sigma Aldrich Quimica), fluconazole ...

Cryptococcus gattii Infections

“…A few studies have examined the roles of the cryptococcal ERG11 gene and of the ATP-binding cassette transporters in resistance in C. neoformans (377)(378)(379)(380); whether these observations are applicable to C. gattii are uncertain. Mutations in, or overexpression of, the ERG11 gene, encoding the azole target lanosterol 14-␣ demethylase, and overexpression of one or more plasma membrane proteins that pump azoles out of the cell are well recognized mechanisms by which fungi acquire resistance to azoles (379).…”

“…The latter isolate contained a point mutation responsible for the amino acid substitution G484S (377). However, the possibility of another mechanism of resistance was not excluded.…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…In C. neoformans, the alteration of the azole target Erg11 has been documented for numerous clinical isolates (492,602). Recently, a three-dimensional structure of C. neoformans Erg11 was elucidated, and residues important for its normal enzymatic function as well as those that interact with fluconazole were identified (531).…”

Resistance to Voriconazole Due to a G448S Substitution in Aspergillus fumigatus in a Patient with Cerebral Aspergillosis

“…The G448S amino acid substitution described here corresponds to the G464S substitution in Candida albicans ERG11 and the G484S substitution in Cryptococcus neoformans, both of which are involved in fluconazole resistance (25,27). Several studies have demonstrated that this amino acid substitution detected in C. albicans ERG11 confers a change in the orientation of the P450 heme-binding domain, leading to decreased azole binding and decreased catalytic activity of the enzyme (15,27).…”